This invention is related to photoactive nucleoside analogs that can be incorporated into synthetic oligonucleotides during automated DNA synthesis for use in crosslinking of complementary target nucleic acid sequences.
The use of crosslinkable probes in nucleic acid hybridization assays to crosslink to target sequences is demonstrated in U.S. Pat. No. 4,826,967 byGlass.; compounds are based on furocoumarin (or psoralen) attached to existing polynucleotides (usually through adduct formation) and are satisfactory for many applications. However, the crosslinking group/nucleoside adduct is difficult to synthesize, particularly in large quantities. In U.S. Pat. No. 5,082,934, Saba et al. describe a photoactivatible nucleoside analog comprising a coumarin moiety linked through its phenyl ring to the 1-position of a ribose or deoxyribose sugar moiety in the absence of an intervening base moiety. The resulting nucleoside analog is used as a photo-crosslinking group when inserted into a polynucleotide as a replacement for one or more of the complementary nucleoside bases present in a probe used in hybridization assays.
Nevertheless, new types of compounds that offer additional advantages, such as stability throughout probe synthesis and use, and conformational flexibility, continue to remain desirable.
The current invention provides non-nucleosidic, stable, photoactive compounds that can be used as photo-crosslinking reagents in nucleic acid hybridization assays and therapeutic applications, as well as techniques and intermediates that can be used to prepare the final products.
The compounds comprise coumarinyl derivatives prepared by linking the phenyl ring of a coumarin molecule or derivative to a hydroxy or polyhydroxy hydrocarbon molecule, such as one of the terminal hydroxy groups of a glycerol molecule. Not to be limited by theory, it is considered that the (poly)hydroxy hydrocarbon moiety of the resulting compound is equivalent to the sugar of a nucleoside, whereas the coumarin moiety occupies the position of a base. Accordingly, the compounds can be inserted into growing polynucleotide chains using automated (or manual) techniques of polynucleotide synthesis. The double bond between the 3- and 4-positions of the coumarin ring system is a photoactive group that covalently crosslinks to nucleosides in the complementary strand when an oligonucleotide containing this non-nucleoside analog (the xe2x80x9cprobexe2x80x9d) is used in a hybridization assay and/or therapeutic application.
For the most part, the photoactive compound has the formula: 
in which the substituents and linking groups are described below in more detail.
The (poly)hydroxy hydrocarbon backbones give maximum flexibility and stability to the oligosaccharide structure in which they are located as well as good solubility in aqueous and organic media.